Project Summary/Abstract Breast cancer (BC) has several distinct molecular subtypes, including estrogen receptor (ESR1) positive and triple negative BC (TNBC). A significant proportion of ESR1-positive therapy sensitive-BCs (TS-BC) initially respond to antiestrogens or aromatase inhibitors, but become therapy resistant-BCs (TR-BC) and progress to incurable metastases. Further, TNBC subtype has a more aggressive clinical course and lack targeted therapies. Development of effective therapies for women with TR-BC and TNBCs represents the highest unmet need. Recent studies revealed the potential role of several members of the Nuclear Receptor (NR) superfamily as molecular drivers in TR-BC and TNBC, including the androgen receptor (AR), glucocorticoid receptor (GR) and the orphan NR tailless (TLX, NR2E1). The specificity and magnitude of NR signaling is mediated by the interaction between NR and critical coregulators and depending on the molecular context in which NRs and NR coregulators are altered, they may contribute to BC progression. The variability of the contribution of specific NRs and NR coregulators to disease progression in TR-BCs and TNBCs poses a therapeutic challenge but also an opportunity for agents that can target multiple NRs and NR coregulators. We have developed a first-in-class polyfunctional small molecules, ERXs that have activity in the TR-BCs and TNBCs. ERXs block NR and coregulator interactions. Uniquely, our preliminary studies identified three lead compounds with differential activity to distinct BC molecular subtypes: ERX-11 (activity against TS-BC, TR-BCs), ERX-41 (activity against TS-BCs, TR-BCs and TNBCs) and ERX-1113 (activity against TNBC only). The objective of this proposal is to further develop lead ERXs to treat TR-BC and TNBC. Our overarching hypothesis is that targeting the NR- coregulator interactome will have therapeutic utility in treating TR-BC and TNBC. Our initial preliminary studies indicated that ERX-11 targets ESR1, while the molecular targets of ERX-41 and ERX-1113 are not yet defined. In Aim1, we will determine the mechanism of action of ERX derivatives using a novel forward genetics approach to identify the molecular target(s) of ERX compounds in ESR1+ BC and TNBCs and establish the molecular interactome using unbiased mass spectroscopy-based approaches and whole genome sequencing approaches. In Aim2 we will optimize the translatability of ERX derivatives and conduct detailed PK, PD, tolerability and toxicity studies on lead ERX compounds. In Aim3, we will test of the efficacy of ERX compounds in using patient derived explant tissues, endocrine resistant models, and by using syngeneic, orthotopic xenografts and in patient derived xenografts (PDX). This proposal is innovative as ERXs block multiple critical protein-protein interactions, and uniquely have activity against a large number of TR-BC and TNBC cell lines. Successful completion of the proposed studies will lead to the development of first-in-class cancer therapy drugs that addresses the critical need of targeting TR-BC and TNBC.